*3.1. The CeCyD Website Allows a Quick Estimation of the Cell Cycle Phases Duration*

Many studies have been using the two formulas developed by Williams (1971) [21] and Stanners and Till (1960) [22] to estimate the length of the cell cycle phases [8,27–32]. However, these estimations demand time and attention due to a large number of calculations involved. Also, they are subject to errors during the calculations. To facilitate the calculations and optimize the time consumed of these estimations, we developed a website called CeCyD (Cell Cycle Duration estimator), as shown in Figure 1A. CeCyD is available at the address http://cecyd.vital.butantan.gov.br/.

Briefly, CeCyD is a user-friendly website able to calculate the values of cytokinesis (C), mitosis (M), G2, S, and G1 phases of the cell cycle, for any organism. For this, the user needs the following experimental parameters: doubling time, percentage of cells in cytokinesis, percentage of cells in mitosis, minimum time to detect two EdU-labeled nuclei in the same cell, percentage of cells EdU-labeled after EdU pulse, and the duration of this EdU pulse.

To test and evaluate the efficiency of the CeCyD, we first obtained the parameters required for *L. major* and *T. cruzi* (CL Brener strain) from experimental analyses, as displayed in Figure S1. Then, we withdrew the same parameters for *T. brucei* from our previous study [8]. Next, we imputed the parameters in the CeCyD and estimated the duration of the cell cycle phases for each of these organisms, as shown in Figure 1B. *T. cruzi* presented G1 = 5.91 h (0.246 ccu), S = 9.86 h (0.411 ccu), and G2 + M + C = 8.23 h (0.343 cuu); *L. major* presented G1 = 5.52 h (0.53 ccu), S = 3.2 h (0.31 ccu), and G2 + M + C = 1.78 h (0.16 ccu); and *T. brucei* presented G1 = 3.37 h (0.397 ccu), S = 2.31 h (0.272 ccu), and G2 + M + C = 2.82 h (0.331 ccu). Of note, ccu means cell cycle unit, where one unit corresponds to the doubling time of each organism.

As expected, for *T. brucei*, the values provided by CeCyD were the same as those obtained in our previous work [8]. For both *L. major* and *T. cruzi*, when we compare the values provided by CeCyD with those obtained from other studies [30,32,33], we can observe similarities among the length of the cell cycle phases when EdU is used to monitor DNA replication [30]. However, when 5-bromo-2′ -deoxyuridine (BrdU) is used to monitor DNA replication instead of EdU, the values obtained shown pronounced differences [33]. As already reported by our group [30], this discrepancy can be explained by the fact that there are differences in the detection of BrdU/EdU incorporation assays, i.e., EdU is more sensitive in monitoring DNA replication than the halogenated thymidine analogs (e.g., BrdU).

For our analyses, we used the S-phase duration from *T. cruzi*, *L. major*, *T. brucei*, *S. cerevisiae*, and *S. pombe*. Of note, for *T. brucei*, *S. cerevisiae,* and *S. pombe*, we did not use the CeCyD because the cell cycle phases duration for these organisms were already available [8,25,26,34,35], as shown in Figure 1B. Also, the cell cycle parameters used here were obtained from epimastigote cells of *T. cruzi*, promastigote cells of *L. major*, procyclic cells of *T. brucei*, mother cells of *S. cerevisiae,* and mitotic cells of *S. pombe*.


**Figure 1.** The user-friendly website CeCyD allows a quick estimation of the cell cycle phases duration for any cell type. (**A**) Screenshot of the CeCyD website showing the parameters to be inserted. This website is available at the address http://cecyd.vital.butantan.gov.br/. (**B**) Estimation of the cell cycle phases lengths (G1, S, and G2+M+C/G2+M) for *T. cruzi*, *L. major*, *T. brucei*, *S. cerevisiae*, and *S. pombe*. For *T. cruzi*, *L. major*, and *T. brucei* from calculations made using the CeCyD website. For *S. cerevisiae* and *S. pombe*, the values were obtained from other studies [25,26,34,35].
